Methods and Compositions for Feeder-Free Pluripotent Stem Cell Media Containing Human Serum

ABSTRACT

The present invention provides compositions and methods for the culture and maintenance of pluripotent stem cells. More particularly, the present invention provides for compositions and methods for culturing, maintaining, growing and stabilizing primate pluripotent stem cells in a feeder-free defined media further comprising human serum, or a soluble attachment component of the human serum, for promoting cell attachment.

ACKNOWLEDGEMENT OF FEDERAL RESEARCH SUPPORT

This invention was made, at least in part, with funding from NCRR(5R24RR021313-05). Accordingly, the United States Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to compositions and methods forculturing pluripotent stem cells and/or cancer stem cells comprising adefined culture medium containing human serum.

2. Background of the Invention

Embryonic stem (ES) cells are a powerful model system for theinvestigation of mechanisms underlying pluripotent cell biology anddifferentiation within the early embryo, as well as providingopportunities for genetic manipulation of mammals and resultantcommercial, medical and agricultural applications. Furthermore,appropriate proliferation and differentiation of ES cells canpotentially be used to generate an unlimited source of cells suited totransplantation for treatment of diseases that result from cell damageor dysfunction. Other pluripotent cells and cell lines including earlyprimitive ectoderm-like (EPL) cells as described in International PatentApplication WO 99/53021, in vivo or in vitro derived ICM/epiblast, invivo or in vitro derived primitive ectoderm, primordial germ cells (EGcells), teratocarcinoma cells (EC cells), and pluripotent cells derivedby dedifferentiation or by nuclear transfer will share some or all ofthese properties and applications. International Patent Application WO97/32033 and U.S. Pat. No. 5,453,357 describe pluripotent cellsincluding cells from species other than rodents. Human ES cells havebeen described in International Patent Application WO 00/27995, and inU.S. Pat. No. 6,200,806, and human EG cells have been described inInternational Patent Application WO 98/43679.

Yet, to date, only several sub-optimal methods for isolating and growingstem cells from primates have been reported. For example, murineembryonic stem cells are maintained in an undifferentiated state usingfeeder-free cultures supplemented with leukemia inhibitory factor (LIF).On the other hand, human embryonic stem cells differentiate when thecells are cultured without a feeder cell layer or conditioned mediumfrom a suitable feeder cell line, even in the presence of LIF. Systemswhich employ feeder cells (or conditioned media from feeder cellcultures) typically use cells from a different species than that of thestem cells being cultivated, e.g., mouse embryonic fibroblasts (MEF)form the feeder layer in most reported undifferentiated growth of humanembryonic stem cells. Moreover, reports of feeder-free systems typicallyrequire the use of conditioned medium from MEF cultures, which does notcure the need for non-xenogeneic products/agents. Even systems thatemploy human feeder cells have the drawback of exposing theundifferentiated cells to undefined culture conditions, and therefore,many stem cell culture conditions are often not reproducible.

Additionally, for most cell therapies, treatment requires extremelylarge quantities of pluripotent stem cells. Based on the cell numbersrequired for islet transplantation using the Edmonton protocol theestimated quantity of differentiated cells required is on the order of10⁹ to 10¹⁰ cells/patient. Further, depending on the hES-deriveddifferentiated target cell, the quantity of undifferentiated hESCs couldbe orders of magnitude higher.

There is a need, therefore, to identify methods and compositions for theculture, stabilization and large-scale production of a uniformpopulation of primate pluripotent stem cells for therapeutic purposes;and wherein the culture compositions are defined and/or produced to GMPstandard.

The present invention described in detail below provides methods andcompositions to culture undifferentiated hESCs using agents which havenot been exposed or derived from non-humans, thus providing a saferculture media.

SUMMARY OF THE INVENTION

The invention provides compositions and methods for a defined media thatsupports the long-term cultivation of undifferentiated stem cells. Themedia is substantially feeder-free (i.e., feeder cells or feedercell-conditioned medium is not required), and substantially free of anymatrix coating.

Thus, in one aspect, the invention concerns defined media useful inculturing stem cells, including undifferentiated pluripotent stem cells.In solution, the media is substantially isotonic as compared to the stemcells being cultured. In a given culture, the particular mediumcomprises a base medium and an amount of various factors necessary tosupport substantially undifferentiated growth of embryonic stem cells.In preferred embodiments, the base medium comprises salts, essentialamino acids, a carbon source that can be metabolized by primate stemcells, and human serum. All these ingredients are supplied in an amountthat will support substantially undifferentiated growth of primate stemcells.

In one embodiment, the invention provides a feeder-free primatepluripotent stem cell tissue culture composition containing anundifferentiated human embryonic stem cell (hESC), a defined culturemedia comprising human serum (hS), wherein the human serum furthercomprises at least one soluble attachment component having at least 100kDa molecular weight, and wherein the composition is essentially free offeeder cells.

In another embodiment, the invention provides a feeder-free primatepluripotent stem cell tissue culture composition containing anundifferentiated human embryonic stem cell (hESC), a defined culturemedia comprising human serum (hS) or a soluble attachment componentthereof wherein the human serum further comprises at least one solubleattachment component having at least 100 kDa molecular weight, andwherein the composition is essentially free of feeder cells.

In still another embodiment, the invention provides a method ofculturing primate embryonic stem cells in a feeder-free defined media byculturing the primate embryonic stem cells in a culture medium which cansupport stem cells, the culture medium being substantially free offeeder cells and further containing about 0.5% to about 2% human serum,the culturing step being conducted for over one month with the embryonicstem cells proliferating in culture while maintaining the potential ofthe stem cells to differentiate into derivatives of endoderm, mesoderm,and ectoderm tissues, and while maintaining the karyotype of theembryonic stem cells.

These and other embodiments of the invention will be described ingreater detail herein.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is therefore anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways.

The phraseology and terminology used herein is for the purpose ofdescription and should not be regarded as limiting. The use of“including”, “comprising”, or “having”, “containing”, “involving”, andvariations thereof herein, is meant to encompass the items listedthereafter and equivalents thereof as well as additional items.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1E show photomicrograph images of pluripotent stem cell culturesin a defined media containing various concentrations of human serum atpassages 0 to 3 (p0 to p3) and days 1 through 4 (d1-d4).

FIG. 2 is a photomicrograph of an agarose gel containing RT-PCR resultsusing OCT4, NANOG, REX1, SOX2, UTF1, CRIPTO, FOXD3, TERT, DPPA5 AFP,MSX1 and HAND1 cell markers of pluripotent hESCs (lanes 2-11, left toright) and differentiated hES-derived cells (lanes 12-14).

FIG. 3 is a graph describing normalized, relative expression of SOX17,HNF1B and HNF4A in pluripotent hESCs (passage 67; lane 1, left), d3definitive endoderm cultures (lanes 2, 4 and 5), and d5 foregut endodermcultures (lanes 3 and 6).

DETAILED DESCRIPTION OF THE INVENTION

Use of human serum as a matrix for the growth of human embryonic stemcells (hESCs) under feeder-free conditions is described by Stojkovic etal. (Stem Cells Express (2005) Stem Cells 23:895-902, originallypublished online May 11, 2005). The method involves coating plates withhuman serum (hS) for 1 hour at room temperature, followed by removal ofexcess serum and drying of plates for 1 hour at room temperature. Thehuman serum was derived from male clotted blood, tested and foundnegative for hepatitis B surface antigen, anti-hepatitis C virus, andanti-HIV HIV/HIV2 by FDA-approved tests. Replating of hESC should alsouse serum precoated plates. In some embodiments, the human serum wasused together with hESC-dF-conditioned media. The conditioned medium wasderived from cultures of fibroblast-like cells, which were derived fromspontaneously differentiated hESCs, as described by Stojkovic et al.

The present invention, described in more detail below, provides forcompositions and methods for culturing, maintaining and growing auniform and undifferentiated primate pluripotent stem cell, and inparticular, hESCs, in a defined media and in the absence of a matrix,feeder layer or plate coating.

DEFINITIONS

Unless otherwise noted, the terms used herein are to be understoodaccording to conventional usage by those of ordinary skill in therelevant art. In addition to the definitions of terms provided below,definitions of common terms in molecular biology may also be found inRieger et al, 1991 Glossary of genetics: classical and molecular, 5thEd., Berlin: Springer-Verlag; and in Current Protocols in MolecularBiology, F. M. Ausubel et al, Eds., Current Protocols, a joint venturebetween Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,(1998 Supplement), or any web based dictionary. It is to be understoodthat as used in the specification and in the claims, “a” or “an” canmean one or more, depending upon the context in which it is used. Thus,for example, reference to “a cell” can mean that at least one cell canbe utilized.

As used herein, the term “contacting” (i.e., contacting a cell e.g., adifferentiable cell, with a compound) is intended to include incubatingthe compound and the cell together in vitro (e.g., adding the compoundto cells in culture). It is understood that the cells contacted with thedefined medium can be further treated with a cell differentiationenvironment to stabilize the cells, or to differentiate the cells.

As used herein, the term “stabilize,” when used in reference to thedifferentiation state of a cell or culture of cells, indicates that thecells will continue to proliferate over multiple passages in culture,and preferably indefinitely in culture, where most, if not all, of thecells in the culture are of the same differentiation state. In addition,when the stabilized cells divide, the division typically yields cells ofthe same cell type or yields cells of the same differentiation state. Astabilized cell or cell population in general, does not furtherdifferentiate or de-differentiate if the cell culture conditions are notaltered and the cells continue to be passaged and are not overgrown. Inone embodiment, the cell that is stabilized is capable of proliferationin the stable state indefinitely, or for at least more than 2 passages.In a more specific embodiment, the cells are stable for more than 3passages, 4 passages, 5 passages, 6 passages, 7 passages, 8 passages, 9passages, more than 10 passages, more than 15 passages, more than 20passages, more than 25 passages, or more than 30 passages. In oneembodiment, the cell is stable for greater than approximately 1 month, 2months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, or 11 months of continuous passaging. In anotherembodiment, the cell is stable for greater than approximately 1 year ofcontinuous passaging. In one embodiment, stem cells are maintained inculture in a pluripotent state by routine passage in the defined mediumuntil it is desired that they be differentiated. As used herein, theterm “proliferate” refers to an increase in the number cells in a cellculture.

Hence, as used herein, the term “growth environment” is an environmentin which stem cells (e.g., primate embryonic stem cells) willproliferate in vitro. Features of the environment include the medium inwhich the cells are cultured, and a supporting structure (such as asubstrate on a solid surface) if present.

A “defined” medium refers to a biochemically defined formulationcomprised solely of the biochemically-defined constituents. A definedmedium may include solely constituents having known chemicalcompositions. A defined medium may also include constituents that arederived from known sources. For example, a defined medium may alsoinclude factors and other compositions secreted from known tissues orcells; however, the defined medium will not include the conditionedmedium from a culture of such cells. Thus, a “defined medium” may, ifindicated, include particular compounds added to form the culturemedium.

As used herein, the term “basal medium” refers to a solution of aminoacids, vitamins, salts, and nutrients that is effective to support thegrowth of cells in culture, although normally these compounds will notsupport cell growth unless supplemented with additional compounds. Thenutrients include a carbon source (e.g., a sugar such as glucose) thatcan be metabolized by the cells, as well as other compounds necessaryfor the cells' survival. These are compounds that the cells themselvescannot synthesize, due to the absence of one or more of the gene(s) thatencode the protein(s) necessary to synthesize the compound (e.g.,essential amino acids) or, with respect to compounds which the cells cansynthesize, because of their particular developmental state the gene(s)encoding the necessary biosynthetic proteins are not being expressed assufficient levels. A number of base media are known in the art ofmammalian cell culture, such as Dulbecco's Modified Eagle Media (DMEM),Knockout-DMEM (KO-DMEM), and DMEM/F12, although any base medium thatsupports the growth of primate embryonic stem cells in a substantiallyundifferentiated state can be employed. A “basal medium” as describedherein also refers to the basal medium described in PCT/US2007/062755,filed Jun. 13, 2007, which is herein incorporated in its entirety.

The basal medium may or may not contain “exogenous insulin or insulinsubstitutes”, which refers to insulin or insulin substitutes that is/arenot intentionally added to the compositions or methods of the presentinvention. Thus, in certain embodiments of the present invention, themethods and compositions are free of insulin or insulin substitutes thatare intentionally supplied. The compositions or methods may, however,not necessarily be free of endogenous insulin. As used herein,“endogenous insulin” indicates that the cultured cells may be producinginsulin of their own accord when cultured according to the methods ofthe present invention. Endogenous insulin also may be used to indicateresidual impurities from the primary cell culture or impurities from thestarting materials. In specific examples, the compositions and methodsof the present contain less than 50, 45, 40, 35, 30, 25, 20, 15, 10, 9,8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.25, 0.1 μg/ml, or substantially noamounts of insulin.

To be clear, the term “insulin” refers to the protein, or variant orfragment thereof that binds to the insulin receptor in normalphysiological concentrations and can induce signaling through theinsulin receptor. The term “insulin” encompasses a protein having thepolypeptide sequence of native human insulin, or of other mammalianinsulin, or of any homologs or variants to these sequences.Additionally, the term insulin encompasses polypeptide fragments thatare capable of binding to the insulin receptor to induce signalingthrough the insulin receptor. The term “insulin substitute” refers toany zinc containing compound that may be used in place of insulin togive substantially similar results as insulin. Examples of insulinsubstitutes include, but are not limited to zinc chloride, zinc nitrate,zinc bromide, and zinc sulfate.

Also to be clear, insulin-like growth factors are not insulinsubstitutes or homologs of insulin, as contemplated in the presentinvention. Accordingly, in another specific embodiment, the compositionsand methods of the present invention comprise the use of at least oneinsulin-like growth factor (IGF) or a variant or a functional fragmentthereof. In another embodiment, the compositions and methods of thepresent invention are free of any exogenous insulin-like growth factors(IGFs). In specific embodiments, the compositions and methods of thepresent invention contain less than 200, 150, 100, 75, 50, 25, 20, 15,10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 ng/ml of IGF-1.

The defined media and/or basal media can include “non-essential aminoacid(s)”, which refers to an amino acid species that need not be addedto a culture medium for a given cell type, typically because the cellsynthesizes, or is capable of synthesizing, the particular amino acidspecies. While differing from species to species, non-essential aminoacids are known to include L-alanine, L-asparagine, L-aspartic acid,L-glutamic acid, glycine, L-proline, and L-serine.

The defined media and/or basal media can also include various growthfactors, hence the term “growth factor”, which may or may not beincluded in the defined media described herein, refers to a substancethat is effective to promote the growth of stem cells and which, unlessadded to the culture medium as a supplement, is not otherwise acomponent of the basal medium. Put another way, a growth factor is amolecule that is not secreted by cells being cultured (including anyfeeder cells, if present) or, if secreted by cells in the culturemedium, is not secreted in an amount sufficient to achieve the resultobtained by adding the growth factor exogenously. Growth factorsinclude, but are not limited to, basic fibroblast growth factor (bFGF),acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF),insulin-like growth factor-I (IGF-I), insulin-like growth factor-II(IGF-II), platelet-derived growth factor-AB (PDGF), and vascularendothelial cell growth factor (VEGF), activin-A, and bone morphogenicproteins (BMPs), cytokines, chemokines, morphogens, neutralizingantibodies, other proteins, and other molecules including but notlimited to heregulin as described in PCT/US07/062,755, which is hereinincorporated by reference in its entirety. The defined media culture asdescribed herein also contains substantially no insulin or lackssubstantial amounts of insulin.

The culture conditions described herein are “isotonic”, which termrefers to a solution having essentially the same tonicity (i.e.,effective osmotic pressure equivalent) as another solution with which itis compared. In the context of cell culture, an “isotonic” medium is onein which cells can be cultured without an appreciable net flow of wateracross the cell membranes.

Also, the culture conditions described herein are solutions having “lowosmotic pressure” which refers to a solution having an osmotic pressureof less than about 300 milli-osmols per kilogram (“mOsm/kg”).

Although, a conditioned medium is not employed in the present invention,as used herein, the phrase “conditioned medium” refers to a growthmedium that is further supplemented with soluble factors derived fromcells cultured in the medium. Techniques for isolating conditionedmedium from a cell culture are well known in the art. As will beappreciated, conditioned medium is preferably essentially cell-free. Inthis context, “essentially cell-free” refers to a conditioned mediumthat contains fewer than about 10%, preferably fewer than about 5%, 1%,0.1%, 0.01%, 0.001%, and 0.0001% than the number of cells per unitvolume, as compared to the culture from which it was separated. However,it is contemplated that the skilled artisan can “substantially remove”one or more detectable components of a conditioned medium. For example,the skilled artisan can remove an amount of the detectable, knowncomponent(s) from the conditioned medium which results in a fractionatedconditioned medium as compared to an unfractionated conditioned medium.Fractionation of a conditioned medium can be performed by any method (orcombination of methods) suitable to remove the detectable component(s),for example, gel filtration chromatography, affinity chromatography,immune precipitation, size-exclusion devices etc.

“Human embryonic stem cells” or “hES cells” or “hESCs” or “stem cells”or “pluripotent stem cells” are cells obtained from an animal (e.g., aprimate, such as a human) embryo. These terms and phrases are equivalentto the phrase, “differentiable cell”. A “differentiable cell” is used todescribe a cell or population of cells that can differentiate into atleast partially mature cells, or that can participate in thedifferentiation of cells, e.g., fuse with other cells, that candifferentiate into at least partially mature cells. As used herein,“partially mature cells” are cells that exhibit at least onecharacteristic of the phenotype, such as morphology or proteinexpression, of a mature cell from the same organ or tissue.

Differentiable cells, as used herein, may be pluripotent, multipotentoligopotent or even unipotent, as defined in detail below. In certainembodiments of the present invention, the differentiable cells arepluripotent differentiable cells. In more specific embodiments, thepluripotent differentiable cells are selected from the group consistingof embryonic stem cells, ICM/epiblast cells, primitive ectoderm cells,primordial germ cells, and teratocarcinoma cells. In one particularembodiment, the differentiable cells are mammalian embryonic stem cells.In a more particular embodiment, the differentiable cells are humanembryonic stem cells.

The invention also contemplates differentiable cells from any sourcewithin an animal, provided the cells are differentiable as definedherein. For example, differentiable cells may be harvested from embryos,or any primordial germ layer therein, from placental or chorion tissue,or from more mature tissue such as adult stem cells including, but notlimited to adipose bone marrow, nervous tissue, mammary tissue, livertissue, pancreas, epithelial, respiratory, gonadal and muscle tissue. Inspecific embodiments, the differentiable cells are embryonic stem cells.In other specific embodiments, the differentiable cells are adult stemcells. In still other specific embodiments, the stem cells areplacental- or chorionic-derived stem cells.

Of course, the invention contemplates using differentiable cells fromany animal capable of generating differentiable cells, e.g., pancreatictype cells such as beta cells. The animals from which the differentiablecells are harvested may be vertebrate or invertebrate, mammalian ornon-mammalian, human or non-human. Examples of animal sources include,but are not limited to, primates, rodents, canines, felines, equines,bovines and porcines.

The differentiable cells of the present invention can be derived usingany method known to those of skill in the art. For example, humanpluripotent cells can be produced using de-differentiation and nucleartransfer methods. Additionally, the human ICM/epiblast cell or theprimitive ectoderm cell used in the present invention can be derived invivo or in vitro. Primitive ectodermal cells may be generated inadherent culture or as cell aggregates in suspension culture, asdescribed in WO 99/53021. Furthermore, the human pluripotent cells canbe passaged using any method known to those of skill in the art,including, manual passaging methods, and bulk passaging methods suchenzymatic or non-enzymatic passaging.

As used herein, the term “differentiate” refers to the production of acell type that is more differentiated than the cell type from which itis derived. The term therefore encompasses cell types that are partiallyand terminally differentiated.

In certain embodiments of the present invention, the term “enriched”refers to a cell culture that contains at least 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% of the desired cell lineage.

As used herein, the term “substantially undifferentiated” cell culturerefers to a population of stem cells containing at least about 50%,preferably at least about 60%, 70%, or 80%, and even more preferably, atleast about 90%, undifferentiated, stem cells. Fluorescence-activatedcell sorting using labeled antibodies or reporter genes/proteins (e.g.,enhanced green fluorescence protein [EGFP]) to one or more markersindicative of a desired undifferentiated state can be used to determinehow many cells of a given stem cell population are undifferentiated. Forpurposes of making this assessment, one or more cell surface markerscorrelated with an undifferentiated state (e.g., SSEA-4, Tra-1-60, andTra-1-81), as well as the typical pluripotent stem cell transcriptionfactor marker, Oct-4, can be detected. Telomerase reverse transcriptase(TERT) activity and alkaline phosphatase can also be assayed. In thecontext of primate stem cells, positive and/or negative selection can beused to detect, for example, by immuno-staining or employing a reportergene (e.g., EGFP), the expression (or lack thereof) of certain markers(e.g., Oct-4, SSEA-4, Tra-1-60, Tra-1-81, SSEA-1, SSEA-3, nestin,telomerase, Myc, p300, and Tip60 histone acetyltransferases, andalkaline phosphatase activity) or the presence of certainpost-translational modifications (e.g., acetylated histones), therebyfacilitating assessment of the state of self-renewal or differentiationof the cells. Also, undifferentiated cells described herein have typicalstem cell morphology which is well described in the art.

“Pluripotent” refers to cells that are capable of differentiating intoone of a plurality of different cell types, although not necessarily allcell types. An exemplary class of pluripotent cells is embryonic stemcells, which are capable of differentiating into any cell type in thehuman body. Thus, it will be recognized that while pluripotent cells candifferentiate into multipotent cells and other more differentiated celltypes, the process of reverse differentiation (i.e., de-differentiation)is likely more complicated and requires “re-programming” the cell tobecome more primitive, meaning that after re-programming, it has thecapacity to differentiate into more or different cell types than waspossible prior to re-programming.

“Totipotent” refers to cells that are capable of differentiating intoany cell type, including pluripotent, multipotent, and fullydifferentiated cells (i.e., cells no longer capable of differentiationinto various cell types), such as, without limitation, embryonic stemcells, neural stem cells, bone marrow stem cells, hematopoietic stemcells, cardiomyocytes, neuron, astrocytes, muscle cells, and connectivetissue cells.

As used herein, “multipotent cells” include cells and their progeny,which may be able to differentiate into, or give rise to, multipotent,oligopotent and unipotent progenitor cells, and/or one or more mature orpartially mature cell types, except that the mature or partially maturecell types derived from multipotent cells are limited to cells of aparticular tissue, organ or organ system. For example, a multipotenthematopoietic progenitor cell and/or its progeny possess the ability todifferentiate into or give rise to one or more types of oligopotentcells, such as myeloid progenitor cells and lymphoid progenitor cells,and also give rise to other mature cellular components normally found inthe blood. “Oligopotent cells” include cells and their progeny whoseability to differentiate into mature or partially mature cells is morerestricted than multipotent cells. Oligopotent cells may, however, stillpossess the ability to differentiate into oligopotent and unipotentcells, and/or one or more mature or partially mature cell types of agiven tissue, organ or organ system. One example of an oligopotent cellis a myeloid progenitor cell, which can ultimately give rise to matureor partially mature erythrocytes, platelets, basophils, eosinophils,neutrophils and monocytes. “Unipotent cells” include cells and theirprogeny that possess the ability to differentiate or give rise to otherunipotent cells and/or one type of mature or partially mature cell type.

Also, a “normal” stem cell refers to a stem cell (or its progeny) thatdoes not exhibit an aberrant phenotype or have an aberrant genotype, andthus can give rise to the full range of cells that can be derived fromsuch a stem cell. In the context of a totipotent stem cell, for example,the cell could give rise to, for example, an entire, normal animal thatis healthy. In contrast, an “abnormal” stem cell refers to a stem cellthat is not normal, due, for example, to one or more mutations orgenetic modifications or pathogens. Thus, abnormal stem cells differfrom normal stem cells.

Again, although the present invention does not employ a tissue culturevessel coating or matrix, e.g., an “extracellular matrix”, an“extracellular matrix” or “matrix” as used herein, refers to one or moresubstances that provide substantially the same conditions for supportingcell growth as provided by an extracellular matrix synthesized by feedercells. The matrix may be provided on a substrate (e.g., fibronectin,collagen, MATRIGEL™, and the like).

As used herein, the term “feeder cells” or “feeder cell layers” arecells which grow in vitro and are co-cultured with a target cell, e.g.,co-cultured with stem cells. As used herein, the term “essentially freeof a feeder cell” or “feeder-free” and equivalents thereof, refer totissue culture conditions that do not contain intentionally added feedercells. Also, a cell culture is “essentially feeder-free” when it doesnot contain exogenously added conditioned medium taken from a culture offeeder cells nor exogenously added feeder cells in the culture, where“no exogenously added feeder cells” means that cells to develop a feedercell layer have not been purposely introduced for that reason, Ofcourse, if the cells to be cultured are derived from a seed culture thatcontained feeder cells, the incidental co-isolation and subsequentintroduction into another culture of some small proportion of thosefeeder cells along with the desired cells (e.g., undifferentiatedprimate stem cells) should not be deemed as an intentional introductionof feeder cells. In such an instance, the culture contains a de minimusnumber of feeder cells. By “de minimus”, it is meant that number offeeder cells that are carried over to the instant culture conditionsfrom previous culture conditions where the differentiable cells may havebeen cultured on feeder cells. Similarly, feeder cells or feeder-likecells that develop from stem cells seeded into the culture shall not bedeemed to have been purposely introduced into the culture.

The present invention contains a defined media further containingvarious amounts of human serum, for example, from about 0.5% to about40%, from about 0.5% to about 30%, from about 0.5% to about 20%, fromabout 0.5% to about 10%, from about 0.5% to about 5%, from about 0.5% toabout 3%, from about 0.5% to about 2%, and from about 0.5% to about 1%.However, typical defined media stem cell cultures use the term“essentially serum-free” which refers to exogenously added serum. Serumadded in such media is typically in greater amounts than that describedherein. Of course, if the cells being cultured produce some or all ofthe components of serum, or if the cells to be cultured are derived froma seed culture grown in a medium that contained serum, the incidentalco-isolation and subsequent introduction into another culture of somesmall amount of serum (e.g., less than about 1%) should not be deemed asan intentional introduction of serum.

As used herein, the term “size-exclusion device” refers to filtrationdevices capable of separating materials on the basis of their size. Onenon-limiting exemplary class of such devices is the filtration-typeclass, wherein sample components are separated on the basis of molecularweight through a matrix that allows small molecules to pass through morerapidly than larger molecules. Typically, filtration-type devices arecharacterized by a molecular weight cutoff that represents an upperlimit of the molecular weight of molecules that are able to pass throughthe matrix. Typically, a sample solution or reaction mixture is forcedthrough the molecular weight separation matrix by application ofcentrifugal force (by centrifugation) or positive pressure (e.g.,application of gaseous pressure or application of a piston above thesolution or reaction mixture). In some embodiments, the matrix isprovided as a membrane. Such devices typically have an integral filtermaterial between an upper and a lower chamber. Examples of suchsize-exclusion devices are the commercially available Microcon 300, 100,50, 30, 3, and Centricon 3K, 30K, 50K, 100K, and 300K (all fromMillipore), and Nanosep available in 10K, 30K, 100K and 300k cutoffs(all from Pall Corp.). The Microcon and Centricon units are the samedevices, but differ in volume of solution that can be used. Alsoavailable is a 96-well plate, for processing many samplessimultaneously. Use of such a multi-sample well plate renders thepurification process less burdensome. Thus, in some embodiments, the“size-exclusion device” comprises a porous, size-discriminating barrierthat allows smaller molecules (e.g., smaller than a predeterminedmolecular weight cutoff as exemplified above) to pass through whileretaining larger molecules (which have molecular weights greater thanthe cutoff). For example, such a device can be an ultrafiltrationdevice, comprising a porous membrane, such as a Microcon or Centriconfiltration device of the type just described. The molecular weightcut-off of the size exclusion device is selected to retain desiredcomponents, and allow undesired species to pass through. Thus, suchembodiments operate by retaining larger molecules which can betransiently trapped in pores of the particles while smaller moleculespass through with bulk eluant, such that smaller molecules elute first.In some embodiments, the size-exclusion device has a 300K cut off or a100K cut off, or a 30K cut off. It will be appreciated, however, thatsize exclusion device having any suitable cut-off can be employed in thepresent methods.

The methods described herein comprise plating the cells in an adherentculture. As used herein, the terms “plated” and “plating” refer to anyprocess that allows a cell to be grown in adherent culture. As usedherein, the term “adherent culture” refers to a cell culture systemwhereby cells are cultured on a solid surface, which may in turn becoated with an insoluble substrate that may in turn be coated withanother surface coat of a substrate, such as those listed below, or anyother chemical or biological material that allows the cells toproliferate or be stabilized in culture. The cells may or may nottightly adhere to the solid surface or to the substrate. The substratefor the adherent culture may comprise any one or combination ofMATRIGEL™ polyornithine, laminin, poly-lysine, purified collagen,gelatin, fibronectin, tenascin, vitronectin, entactin, heparin sulfateproteoglycans, poly glycolytic acid (PGA), poly lactic acid (PLA), andpoly lactic-glycolic acid (PLGA). Furthermore, the substrate for theadherent culture may comprise the matrix laid down by a feeder layer, orlaid down by the pluripotent human cell or cell culture.

As used herein, the phrase “soluble attachment component” or “solubleattachment agent” or equivalents thereof, refers to a soluble component,e.g., a polypeptide, contained in human serum which functions to promotepluripotent stem cell attachment to a substrate, e.g., to promotepluripotent stem cells to attach to plastic tissue culture vessel. Thesoluble attachment component is at least 100 kDa, at least 150 kDa, atleast 200 kDa, at least 250 kDa, or at least 300 kDa or higher, andremains in suspension or is a solute in solution. That is, such asoluble attachment component is not provided for coating of tissueculture vessel and therefore does not form the basis of a matrix coatingas typically understood in primate stem cell cultures.

As used herein, the term, “tissue culture vessel” or “tissue cultureplastic” or equivalents thereof refers to any and all containers, nowknown or later discovered, commonly used to culture and grow stem cells.For example, some tissue culture vessels have a base with a bottom walland a plurality of sidewalls extending up from said bottom wall, a coverextending across said sidewalls and opposed to the bottom wall.Alternatively, the cover can be formed with at least one opening and aseptum extending across said opening for permitting access to interiorportions of said vessel by another device. Still, many tissue culturevessels are of generally prismatic shape (e.g., bioreactors) with aplurality of upstanding sidewalls extending between opposed top andbottom walls. The sidewalls generally are constructed so that the lengthand width of the vessel exceed the height. As a result, the bottom wallof the vessel defines a fairly large surface area relative to the volumeof the vessel. A tubular neck typically is formed at one of thesidewalls of the vessel to provide access to the interior. The outersurface of the neck may be formed with an array of threads for receivinga cap. Thus, one skilled in the art will recognize that the presentinvention and use of media containing hS to promote cell attachment tothe base, bottom or sidewalls of a tissue culture or tissue culture-likevessel, encompasses many and potentially all tissue culture vesselsindependent of shape, size and volume.

The present invention may also be understood more readily by referenceto the following detailed description of the preferred embodiments ofthe invention and the Examples included herein. However, before thepresent compositions and methods are disclosed and described, it is tobe understood that this invention is not limited to specific nucleicacids, specific polypeptides, specific cell types, specific host cells,specific conditions, or specific methods, etc., as such may, of course,vary, and the numerous modifications and variations therein will beapparent to those skilled in the art.

Defined Culture for Growth, Proliferation and Maintenance of PluripotentStem Cells

The present invention describes use of methods and compositions for adefined media, which was first described in PCT/US2007/062755, filedJun. 13, 2007, which is herein incorporated in its entirety.

In guiding hESC technology toward the clinic, one key issue to beaddressed is a lack of standardization in the culture and maintenance ofhESCs. In the absence of mouse embryonic fibroblast (MEF) feeder layers,many researchers rely on “conditioning” in which medium is first exposedto MEFs to acquire soluble factors that support the propagation ofundifferentiated hESCs in culture. It has been difficult to discern howMEF conditioning enables hESCs to maintain an undifferentiated state.Other common features of more recently developed hESC culture conditionsinclude the presence of FGF2, the absence of serum, and the presence ofa serum substitute such as KnockOut Serum Replacer (KSR-Invitrogen,proprietary formulation). Other factors suggested to play a role insupporting the maintenance of hESCs include TGFβ1, activin A (ActA),PDGF and sphingosine-1-phosphate, BIO, a small molecule inhibitor ofGSK3β, and neurotrophins. Several defined medium systems have beendescribed for hESCs and are based upon FGF2 in combination with nodal,TGFβ1, GABA, pipecolic acid, plus lithium chloride, Wnt3a plusApril/BAFF, or the N2/B27 supplements. While these studies have focusedon identifying growth factors and conditions that support theproliferation of undifferentiated hESCs, little is known about the cellsurface receptors that are activated when hESCs are exposed toconditions favorable for self-renewal.

The methods and compositions of a defined media which supports hESCself-renewal is described in PCT/US2007/062755 and incorporated hereinby reference in its entirety. A benefit of using a defined media is thatthe ingredients comprising the media are known and have knownquantities. In contrast an undefined medium has some complexingredients, consisting of a mixture of many, many chemical species inunknown proportions. The reasons for utilizing chemically defined mediaare also pragmatic because such media is reproducible at different timesand in different laboratories. Defined media can be varied in acontrolled manner and, are free of unknown biological activities, suchas enzymes and, alternatively, growth factors, which may affect theresponses being studied.

The compositions and methods of the present invention are useful forculturing cells, in particular, differentiable cells. It is understoodthat at different points during culturing the differentiable cells,various components may be added to the cell culture such that the mediumcan contain components other than those described herein.

The compositions and methods comprise a basal salt nutrient solution. Asused herein, and as described in PCT/US2007/062755, which is hereinincorporated in its entirety, a basal salt nutrient solution refers to amixture of salts that provide cells with water and certain bulkinorganic ions essential for normal cell metabolism, maintain intra- andextra-cellular osmotic balance, provide a carbohydrate as an energysource, and provide a buffering system to maintain the medium within thephysiological pH range. Examples of basal salt nutrient solutionsinclude, but are not limited to, Dulbecco's Modified Eagle's Medium(DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPM11640, Hams F-10, Ham's F-12, β-Minimal Essential Medium (βMEM),Glasgow's Minimal Essential Medium (G-MEM), and Iscove's ModifiedDulbecco's Medium, and mixtures thereof. In one particular embodiment,the basal salt nutrient solution is an approximately 50:50 mixture ofDMEM and Ham's F12.

In one embodiment, the compositions and methods of the present inventionprovide for a soluble attachment factor or agent, such solubleattachment components is contained in the human serum, which at theappropriate concentration range facilitates hESC cell attachment totissue culture type plastic. Such cell attachment allows cells to attachand form a monolayer but in the absence of a feeder layer or a substratecoating, e.g., a matrix coating, MATRIGEL™ and the like. Preferably,human serum is utilized in order to provide an animal-free environment.Human serum can be obtained from any commercial supplier of tissueculture products, examples include Gibco-Invitrogen Corporation (GrandIsland, N.Y. USA), Sigma (St. Louis Mo., USA) and the ATCC (Manassas,Va. USA). The serum used in the present invention is provided at aconcentration range of about 0.1% to about 20%, more preferably about0.1 to about 3%, more preferably about 0.5 to about 2%, more preferablyabout 0.5 to about 1.5%, and more preferably about 0.5 to about 1%.

In another aspect of the invention, the human serum is fractionatedusing microfluidic size-exclusion devices. Various microfluidic devicesfor filtration are commercially available. The devices can be used, forexample, for gel filtration, size-exclusion filtration, ion-exchangefiltration, or combinations of these filtration techniques. For example,filtration materials can be loaded and/or included in the devices, andcan include small beads of filtration materials. Size-exclusionmaterials can be used that can retain smaller molecules of an aqueoussample while allowing larger molecules of the sample to pass through oraround. For example, P-10BIO-GEL materials from Bio-Rad can be used andare composed of acrylamide particles that are have 45-100 μm in averageparticle size diameter. Samples can be manipulated through themicrofluidic devices via gravity pressure differentials, or centripetalforce, for example. The resulting filtrates that elute from the devicescan then be analyzed, used, or subsequently passed on through the deviceto a subsequent stage of processing. In one aspect of the invention, thesize-exclusion device has a 300 KDa cut off, or a 100 KDa cut off, or a30 KDa cut off spin columns, e.g., Microcon spin columns.

According to other aspects of the invention, the filter fit material canbe “press fit” into the respective chamber, placed in the respectivechamber, or otherwise positioned in the respective chamber.

In another aspect of the invention, the gel filtration material can bedisposed in a channel of the microfluidic device. The gel filtrationmaterial can be loaded into the device by pipetting into an inputopening of the device and/or drawing the material into the device byusing vacuum force, for example, applied to an output opening of thedevice. A channel of the device can be filled with a gel filtrationmaterial by pressure loading the gel filtration material through aninput opening of the device to dispense the gel filtration material in achannel or chamber of the device. Once the channel is filled withhydrated gel filtration material, the device can be centrifuged tode-water the gel filtration material and to “pack” the gel filtrationmaterial, forming a purification column. This process can be used toprepare the device for sample filtration and can be used to removeunnecessary or excess water or buffer from the gel filtration material.

Thus, it will be appreciated that microfluidic size exclusion devices,in general, having any suitable filtration material and/or agent andhaving any suitable molecular weight cut-off can be employed in thepresent methods; and the compositions and methods described herein arenot limited to the described devices.

Compositions and methods of the invention can also include at least oneactivator of an FGF receptor, including any of the FGF polypeptides,functional fragments thereof or variants thereof. Compositions andmethods of the invention can include serum albumin (SA), e.g., bovine SA(BSA) or human SA (HAS), at least one insoluble substrate. For example,the differentiable cells may be placed on a cell culture surface thatcomprises such compounds as, but not limited to, polystyrene andpolypropylene.

The present invention is distinguished from other defined cultureconditions in that the compositions and methods comprise proliferatingpluripotent stem cells substantially free of feeder cells or layers, or“feeder-free”, or a conditioned medium produced by collecting mediumfrom a culture of feeder cells. In addition, the compositions andmethods of the present invention do not require for the pluripotent stemcells to proliferate and grow on any substrate, including any substratecoated with extracellular matrix components (i.e., collagen, laminin,fibronectin, proteoglycan, entactin, heparan sulfate, and the like,alone or in various combinations), or MATRIGEL™. The concentration ofhuman serum provided herein is sufficient to promote cell attachment andpromote growth and proliferation of undifferentiated pluripotent stemcells.

In one embodiment, differentiable cells are contacted with at least oneof the compositions of the invention in the absence a feeder cell layer,such that the cells are maintained in an undifferentiated state for atleast one (1) to twelve (12) months or more. Pluripotency can bedetermined through characterization of the cells with respect to surfacemarkers, transcriptional markers, karyotype, and ability todifferentiate to cells of the three germ layers. These characteristicsare well known to those of ordinary skill in the art.

It is contemplated that the differentiable cells can be passaged usingenzymatic, non-enzymatic, or manual dissociation methods prior to and/orafter contact with the defined medium of the invention. Non-limitingexamples of enzymatic dissociation methods include the use of proteasessuch as trypsin, collagenase, dispase, and ACCUTASE™. In one embodimentACCUTASE™ is used to passage the contacted cells. When enzymaticpassaging methods are used, the resultant culture can comprise a mixtureof singlets, doublets, triplets, and clumps of cells that vary in sizedepending on the enzyme used. A non-limiting example of a non-enzymaticdissociation method is a cell dispersal buffer. Manual passagingtechniques have been well described in the art, such as in Schulz etal., 2004 Stem Cells, 22(7):1218-38. The choice of passaging method isinfluenced by other culture conditions, including but not limited tofeeders and/or extracellular matrices; although neither is anticipatedin the present invention.

The disaggregation solution used in the methods of the present inventioncan be any disaggregation solution capable of breaking apart ordisaggregating the cells into single cells, without causing extensivetoxicity to the cells. Examples of disaggregation solutions include, butare not limited to, trypsin, ACCUTASE™, 0.25% Trypsin/EDTA, TrypLE, orVERSENE™ (EDTA) and trypsin. The methods of the present invention neednot result in every cell of the confluent layer being disaggregated intosingle cells, provided that at least a few single cells aredisaggregated and capable of being re-cultured.

The compositions and methods of the invention may contain virtually anycombination of the components set out above or described elsewhereherein, e.g. PCT/US2007/062755. As one skilled in the art wouldrecognize, the components of the compositions and methods of theinvention will vary according to the protocol design. Accordingly, oneembodiment of the present invention relates to culturing differentiablecells in 96-well plates and/or 384-well plates, or in larger vesselsincluding, bioreactors, cell factories, or other automated, or GMPcompliant, specialized devices available or devised by one skilled inthe art. Thus, the methods of the present invention are not limited tospecific culture chamber dimensions.

The culture methods of the invention comprise culturing stem cells suchas primate embryonic stem cells in a growth environment that isessentially feeder-free and which comprises a defined, isotonic culturemedium according to the invention and in the absence of a matrix. Suchdefined, isotonic culture media contain the essential components thatare required for maintaining the stem cells (e.g., pluripotent stemcells) in a substantially undifferentiated state (or their functionalequivalents). The cells can be cultured in such an environment in anysuitable culture vessel under conditions that allow an undifferentiatedstate to be maintained.

Using such methods, populations of stem cells, including substantiallyundifferentiated primate stem cells, e.g., human embryonic stem cells,can be isolated from the resulting cell cultures, thereby representinganother aspect of the invention. Such populations can be isolated by anysuitable technique. Such techniques include affinity chromatography,panning, and fluorescence-assisted cell sorting. Such techniques eachemploy one or more separation reagents (for example, but not restrictedto, antibodies and antibody fragments, reporter genes/proteins, etc.)that are specific for a cell-based marker indicative of anundifferentiated state. In the context of substantially undifferentiatedhuman embryonic stem cells, such markers include, for example, but arenot restricted to the transcriptional factor Oct4, and cell surfacemarkers SSEA-4, Tra-1-60, and Tra-1-81. Other markers includetelomerase, Myc, p300, and Tip60 histone acetyltransferases, acetylatedhistones, and alkaline phosphatase and typical stem cell morphology.

Method for Identifying Factors/Agents Capable of Promoting PluripotentStem Cell Growth And/or Differentiation

Another embodiment of the invention relates to a method of using stemcells to identify factors that promote the cells' differentiation, or,alternatively, the cells' continued maintenance and stabilization in asubstantially undifferentiated state. Briefly, in the context ofdifferentiation or maintenance of a substantially undifferentiatedstate, such methods involve, for example, exposing a test compound tosubstantially undifferentiated primate embryonic stem cells that arebeing cultured in a defined, isotonic culture medium of the invention.Following exposure to the test compound, the cells are assessed todetermine if they have been better maintained in a substantiallyundifferentiated state or induced to differentiate. If the cells havebeen better maintained in a substantially undifferentiated state, thetest compound can be identified as one that promotes an undifferentiatedstate or self-renewal of primate pluripotent stem cells. If the cellshave been induced to differentiate, the test compound can be identifiedas one that promotes differentiation of substantially undifferentiatedprimate pluripotent stem cells. The differentiating cells may befollowed to determine their developmental fate, in other words, todetermine what cell lineage they become as a result of differentiating.In the context of de-differentiation, cells of a more differentiatedstate are exposed to one or more compounds and then assessed todetermine if the exposure resulted in cells of a more primitive type(e.g., a pluripotent stem cell) than those initially exposed to the testcompound. If so, the compound that produces the effect is identified asone that promotes de-differentiation, or reprogramming, of cells.Preferably, these and other screening methods according to the inventionare conducted in a high throughput manner, such that numerous compoundscan be simultaneously screened.

The cell types that differentiate from differentiable cells have severaluses in various fields of research and development including but notlimited to drug discovery, drug development and testing, toxicology,production of cells for therapeutic purposes as well as basic scienceresearch. These cell types express molecules that are of interest in awide range of research fields. These include the molecules known to berequired for the functioning of the various cell types as described instandard reference texts. These molecules include, but are not limitedto, cytokines, growth factors, cytokine receptors, extracellular matrix,transcription factors, secreted polypeptides and other molecules, andgrowth factor receptors.

It is to be understood that one of the benefits provided by culturingstem cells by methods described in the present invention is thereduction and avoidance of contamination of the stem cells withpathogens or antigens foreign to the stem cells. This type ofcontamination can occur when feeder cells from a different species orfrom a different and unrelated individual of the same species are used.The methods of the invention are intended to avoid as much as possiblefurther testing of the stem cell lines (including testing for pathogencontent). In some embodiments, it is anticipated that the human stemcells and/or their differentiated progeny can be transplanted into agenetically related individual without prior testing for some or allpathogen content.

The cell lines may be tested prior to or after cryopreservation fortheir genotype and histocompatibility haplotype, as appropriate.Genotype testing refers to determining the genotype of the stem celllines at one or more resolution levels. It is not necessary to determinethe genotype of the cell line at single nucleotide resolution. Rather,the genotyping must only be carried out at a resolution level thatallows one of ordinary skill to determine the similarity between thestem cell line and any intended recipient thereof. Genotyping can becarried out in a number of ways including but not limited to restrictionfragment length polymorphism (RFLP), single nucleotide polymorphisms(SNPs) and the like.

The cell lines and/or their progeny can also be tested for theirhistocompatibility haplotype; or to determine if they are “normal” or“abnormal” stem cells. A histocompatibility haplotype is a set ofalleles at the histocompatibility gene loci that is used by the immunesystem to distinguish between self and non-self (i.e., foreign) tissuesand/or cells. In humans, the major histocompatibility (MHC) locus iscomposed of four loci on the short arm of chromosome 6. Humans also havea set of minor histocompatibility loci. As an example, human leukocyteantigen (HLA) typing is commonly performed for various transplants suchas hematopoietic cell transplants. Major and minor histocompatibilityantigens are present on cell surfaces and are recognized by the immunesystem as an indicator of the origin of the cell or tissue. Cells ortissues that are viewed as foreign will usually be rejected by therecipient via a host versus graft immune response. However, it issometimes possible to overcome some differences in histocompatibility,particularly those in the minor histocompatibility loci, using forexample immunosuppressive drugs such as but not limited to cyclosporinA, FK506, rapamycin, cyclophosphamide (CY), procarbazine (PCB), andantithymocyte globulin (ATG). Additionally, certain tissues are lesssusceptible to differences at for example the HLA loci in humans. Thesetissues include but are not limited to liver, kidney, and the centralnervous system. It has recently been reported that embryonic stem cellspossess immune privileged properties (Li et al. Stem Cells 200433:448-456).

Kits

The present invention also relates to kits, wherein the kit comprises abasal salt nutrient solution and at least one compound capable ofstimulating ErbB2-directed tyrosine kinase activity. In one embodiment,the kits comprise at least one ErbB3 ligand, as described herein. Inanother embodiment, the kits comprise more than one ErbB3 ligand. Inanother embodiment, the kits comprise at least one of TGF-β or a TGF-βfamily member or a variant or functional fragment thereof as describedherein. In yet another embodiment, the kits comprise more than one ofTGF-β or a TGF-βfamily member or a variant or functional fragmentthereof. In still another embodiment the kits comprise at least onefibroblast growth factor or variant or functional fragment thereof. Inanother embodiment, the kits comprise more than one fibroblast growthfactor or variant or functional fragment thereof. In a specificembodiment, the kits comprise at least one of FGF-7, FGF-10, FGF-22 orvariants or functional fragments thereof. In another embodiment, thekits comprise serum albumin. In still another embodiment, the kitscomprise serum and/or at least one insoluble substrate as describedherein and/or at least one disaggregation solution.

The kits of the invention may contain virtually any combination of thecomponents set out above or described elsewhere herein, which includesbut is not limited to the described defined media culture comprisinghuman serum or a high molecular weight fraction thereof. As one skilledin the art would recognize, the components supplied with kits of theinvention will vary with the intended use for the kits. Thus, kits maybe designed to perform various functions set out in this application andthe components of such kits will vary accordingly.

Throughout this application, various publications are referenced. Thedisclosures of all of these publications and those references citedwithin those publications in their entireties are hereby incorporated byreference into this application in their entirety in order to more fullydescribe the state of the art to which this invention pertains.

EXAMPLES Example 1 Proliferation of Undifferentiated Pluripotent StemCells in a Feeder-Free Culture Containing Small Quantities of HumanSerum

To elucidate a media and cell culture condition that is compatible withthe expected regulatory guidelines governing clinical safety andefficacy, Applicants have provided a hESC scale-up strategy whichfacilitates this requirement as described in more detail below.Long-term cultivation of undifferentiated hESCs in a feeder-free,conditioned-medium-free, and matrix-free medium is crucial for providingan unlimited supply of cells for cell-based therapies, as well as fordirecting the lineage-specific differentiation of hESCs.

Previously, the minimal essential conditions needed to support thelong-term growth of undifferentiated hESCs were determined. SeePCT/US2007/062755). The developmental stage of the stem cells wasassessed, in part, based on morphological analysis, as well as byexpression of cell surface markers and alkaline phosphatase analysis.

In order to establish a xeno-free, feeder-free, conditioned-medium-free,and matrix-free growth culture environment, a culture systemincorporating the defined media described herein and inPCT/US2007/062755 was used. In addition, to maintain culture conditionswhich are free of non-human animal products, but which facilitated cellbinding or attaching to or laying down on the plastic and growth, humanserum was used. The NIH-registered BG01, BG02 and BG03 hESC lines, aswell as CyT49, a hESC line isolated using human feeder cells under GMPconditions (Novocell, San Diego, Calif.) were cultured in the definedmedia including about 0.5 to 2% human serum (hS) for about 12 to 24hours at 37° C. Most commercially available lots of human serum can beused, for example, ALBUMARC human serum was employed for certain hESCcultures described herein. ALBUMARC is a registered name for human serumfrom the American Red Cross Blood Services. It is noteworthy, that theundifferentiated hESCs were grown and maintained in a non-coated tissueculture vessel. The amount of hS utilized promotes cell attachment totissue culture plastic such that coating of the tissue culture vessel,for example, with ECM, fibronectin, collagen and/or MATRIGEL™ and thelike is not required. Also increased concentrations of hS can beemployed, for example up to 20% of hS has been used to culture andmaintain hESC cultures.

The elimination of a matrix requirement provides for a more efficientculture and growth of the cells. Cell cultures were routinely passagedwith ACCUTASE. After seeding in the tissue culture plate for about 12-24hours, the hESCs appeared morphologically normal. Also, karyotypeanalyses of hESCs grown in the defined media and hS were performed usingstandard G-banding techniques.

To determine if the hS was heat sensitive or heat labile, and whether iteffected hESC growth, the hS was first heated to about 50-60° C. forabout 20-60 minutes and then added to the defined media at aconcentration of about 0.5 to 2%. hESCs in this medium did notproliferate significantly, and most died after about 1-3 dayspost-seeding. Hence, a cell attachment promoting component of the hS isheat sensitive and at about 50-60° C., activity decreased significantlyand hESCs did not survive. The hESCs likely did not survive due to theheat sensitivity of one or more soluble attachment factors in the hS,which can become heat-inactivated. Heat-inactivation of these solubleattachment factor(s) prevents pluripotent stem cell attachment to theplastic, which attachment promotes growth and maintenance of the hESCs.

To further determine the approximate size of these soluble attachmentfactors, it was postulated that specific fractions in the hS wereresponsible for potentiating growth and proliferation of the hESCs. Todetermine which fractions, the hS was fractionated using varioussize-exclusion devices, e.g., Microcon 300K, 100K and 30K cut-off spincolumns. The use of these cut-off spin columns allows certain molecularweight proteins to pass through and be collected as the eluant orretentate fraction. hESCs were then cultured in the defined media,containing one of various retentate fractions substantially as describedabove. Retentate fractions from the 300K and 100K cut-off spin columnsappeared to contain particular soluble attachment factors and/or agentswhich promoted undifferentiated hESC growth and proliferation.Undifferentiated hESC growth and proliferation was consistent withvarious retentate fractions from the 300K and 100K cut-off spin columns,and was independent of the commercial source of the (non-heat treated)hS. Thus, those soluble attachment factors capable of being retained inthe retentate fraction in 300K to 100K cut-off spin columns, wereimportant and capable of promoting growth and proliferation.

FIGS. 1A-E are images of hESCs (BG02 cells) growing in defined media(DC-HAIF) on uncoated tissue culture vessels with different amounts ofhuman serum. FIG. 1A is an image of hESCs after 4 days of growth inDC-HAIF containing 1% whole serum on uncoated tissue culture vessels.The cells were incubated with DC-HAIF plus 1% hS for about 24 hours andthen the media was replaced with DC-HAIF. FIG. 1B is a hESC image after1 day of growth in DC-HAIF containing 1% of hS which was partiallyfractionated (retentate fraction was used) on a 300K cut-off spincolumn. FIG. 1C is an hESC image after 4 days of growth in DC-HAIFcontaining about 0.7% hS which was partially fractionated (retentatefraction was used) on a 100K cut-off spin column. FIG. 1D is a hESCimage after 1 passage and after 3 days of growth in DC-HAIF mediacontaining about 0.7% hS which was partially fractionated (retentatefraction) on a 100K cut-off spin column. FIG. 1E is a hESC image of aserially passaged (2 passages) population cultured in DC-HAIF and about1% hS which was partially fractionated (retentate fraction) on a 100Kcut-off spin column.

Additionally, to determine whether stem cell morphology was effectedupon serial passaging of the hESCs under these culture conditions, theundifferentiated hESCs were serially passaged for about 2, 3, 4, 5, 6,7, 8, 9, and 10 times and the like. Undifferentiated hESCs split tosingle cells can also grow and proliferate substantially as described.With an aid of a microscope, all cultures appeared morphologicallynormal. In contrast, when the undifferentiated hESCs were seriallypassaged using whole hS, there was some deterioration in themorphological quality of the hESC cultures as compared to hESCs passagedusing retentate fractions as described above. However, to restoretypical hESC cellular morphology, the hS can be partially purified byfiltering about 20% hS using a 100K cut-off spin column, the collectedeluant/retentate fraction then diluted back to the original volume.Thus, fractionating the hS as described herein largely avoideddeterioration in morphological quality of hESC cultures with serialpassaging. This was also observed with hS concentrations between about0.5 to 1.5%.

To demonstrate the self-renewal of undifferentiated hESCs maintainedunder the above-described defined biologics-free culture conditions,hESCs were enzymatically treated, dissociated into a single cellsuspension, and then cultivated under the defined conditionssubstantially as described above. Undifferentiated mature-sizedsingle-cell-derived hESC colonies began to appear after about 4, 5, 6,and 7 days in vitro.

The plating efficiency was comparable to that observed in hESCs culturedin DC-HAIF as described in PCT/US2007/062755 or cultured in DC-HAIFmedia using MATRIGEL™ as an ECM. The plating efficiency using MATRIGEL™was significantly higher than using MEF-CM or feeders. One explanationis that the dissociated single cells seeded efficiently in hS containingDC-HAIF or defined hESC media as compared to other feeder and or feederand matrix type cultures.

This example identifies the minimal essential components necessary tomaintain primate embryonic stem cells, in particular hESCs, in ahealthy, undifferentiated state capable of both prolonged propagationand then directed differentiation. Having discerned these molecularrequirements, it became possible to derive conditions that would permitthe substitution of poorly-characterized and unspecified biologicaladditives and substrates (including those derived from animals) withentirely defined constituents. This defined culture system has theadvantage of allowing hESCs to be expanded efficiently and stablyfollowing enzyme-mediated dissociation. Therefore, this study provides aviable approach for providing a large supply of well-characterized,clinically-acceptable, healthy cells for cell-based therapies. Inaddition, having established individual components required for theundifferentiated growth of hESCs, it is now possible to assess moreaccurately the effects of other growth factors and compounds on thedevelopmental fate of hESCs. As will be appreciated, defined media arecrucial for directing a requisite number of pluripotent hESCsefficiently, uniformly, stably, and reproducibly towards a specificlineage for therapeutic purposes. Also, FISH analyses of BG02 cellsmaintained for 10 passages in DC-HAIF with 1% 100K human serum fractionexhibited normal counts for human Chromosome 12 (hChr 12; 98% two copy,n=591) and hChr 17 (98% two copy, n=578). Hence, self-renewal ofpluripotent hESCs in media containing a small percentage of human serumdoes not select for the aneuploidies typically observed in hESCs.

Example 2 Long Term-Self Renewal of hESCs Using 100K Human SerumFraction

To be useful, any hESC culture medium has to be able to supportlong-term or prolonged self-renewal of the cells, i.e. for severalpassages. To demonstrate that the DC-HAIF containing hS media describedherein can support long-term or prolonged self-renewal of hESCs, hESCswere cultured in DC-HAIF with about 1% 100k human serum retentatefraction. To make certain that the hESCs maintained their pluripotencyand did not spontaneously differentiate to different cell lineages,RT-PCR analysis was performed using cell markers well known in the artfor describing and identifying pluripotent stem cells and/ordifferentiated stem cell-derived lineages. RT-PCR was performed on hESCcultures after 6 and 10 passages. In control cultures, RT-PCR wasperformed after 53 passages in defined media alone (FIG. 2). FIG. 2shows that typical pluripotent stem cell markers were expressed incultures from 6 (middle panel), 10 (bottom panel) and 53 (top panel)passages, for example, expression of OCT4, NANOG, REX1, SOX2, UTF1,CRIPTO, FOXD3, TERT and DPPA5. In contrast, markers typical ofdifferentiated stem cell-derived lineages, for example, α-fetoprotein(AFP), MSX1, HAND1 were not detected in these cultures. Thus, these dataindicate that hESCs grown in the DC-HAIF with human serum media promotelong term or prolonged self-renewal of pluripotent hESCs and notspontaneous differentiation into hES-derived cell lineages.

Example 3 HESCs Maintained in a Defined Media Containing 100K HumanSerum Fraction can Differentiate into Various Cell Lineages

To determine whether hESCs cultured as described herein could alsodifferentiate to various hES-derived cell lineages, BG02 cells weredifferentiated to hES-derived cells in vitro (FIG. 3). hESCs were firstmaintained for 3 splits for 5 to 7 days in 0.7% or 1% hS fractionatedusing a 100K cut-off spin column. hESCs were then induced todifferentiate on days 5 and 7, respectively, to definitive endoderm byexposing cultures to RPMI containing 2% fatty acid-free BSA, 25 ng/mlWnt3a, 100 ng/ml Activin A and 8 ng/ml FGF2 for 24 hours, followed byRPMI containing 2% BSA, 100 ng/ml Activin A and 8 ng/ml FGF2 for twomore days (d3 of differentiation). Further differentiation of thedefinitive endoderm cells to foregut endoderm was performed by exposingthe cells to RPMI containing 2% BSA, 50 ng/ml FGF7 and 0.25 μMKAAD-cyclopamine for about 2-3 more days (d6 of differentiation). Thecell cultures were examined by qPCR as described previously in D'Amouret al 2005, Nat Biotechnol. 23:1534-1541 and D'Amour et al 2006, NatBiotechnol. 24:1392-1401, which are herein incorporated by reference intheir entirety.

The hES-derived cells (definitive endoderm) were examined on the thirdday of differentiation. FIG. 3 shows graphs describing normalizedexpression levels of various cell markers at d3 (definitive endoderm,DE) and d5 (foregut endoderm, FG). The top panel of FIG. 3 shows thatSOX17 expression is increased significantly at d3 (definitive endoderm);whereas expression levels of HNF1B and HNF4A were unregulated in d5(foregut) samples. Expression levels of SOX17, HNF1B and HNF4A indefinitive endoderm and foregut type hES-derived cells is consistentwith that described in D'Amour et al. 2005 and 2006, supra.

These data demonstrate that the described feeder-free culturesmaintained using retentate fractions from 100K human serum fraction candifferentiate effectively to various hES-derived cell lineages includingdefinitive endoderm and foregut endoderm.

All patents and patent applications, publications, scientific articles,and other referenced materials mentioned in this specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains, and each of which is hereby incorporated byreference to the same extent as if each reference had been incorporatedby reference in its entirety individually.

Applicants reserve the right to physically incorporate into thisspecification any and all materials and information from any suchpatents and patent applications, publications, scientific articles,electronically available information, and other referenced materials ordocuments.

The methods, compositions, and devices described herein are presentlyrepresentative of preferred embodiments and are exemplary and are notintended as limitations on the scope of the invention. Changes thereinand other uses will occur to those skilled in the art which areencompassed within the spirit of the invention and are defined by thescope of the disclosure. Accordingly, it will be apparent to one skilledin the art that varying substitutions and modifications may be made tothe invention disclosed herein without departing from the scope andspirit of the invention.

As used in the claims below and throughout this disclosure, the phrase“consisting essentially of” is meant to include any elements listedafter the phrase, and also may include other elements that do notinterfere with or contribute to the activity or action specified in thedisclosure for the listed elements. Thus, the phrase “consistingessentially of” indicates that the listed elements are required ormandatory, but that other elements are optional and may or may not bepresent depending upon whether or not they affect the activity or actionof the listed elements.

1. A feeder-free primate pluripotent stem cell tissue culturecomposition comprising: a) an undifferentiated human embryonic stem cell(hESC); b) a defined culture media comprising human serum (hS) or asoluble attachment component thereof, wherein the human serum furthercomprises at least one soluble attachment component having at least 100kDa molecular weight, and wherein the composition is essentially free offeeder cells.
 2. The composition of claim 1, wherein the human serum isin a concentration of about 0.5 to about 2% of the media.
 3. Thecomposition of claim 1, wherein the size of the soluble attachmentcomponent is selected from the group consisting of at least 100 kDa, atleast 150 kDa, at least 200 kDa, at least 250 kDa, and at least 300 kDa.4. The composition of claim 1, wherein the component promotes attachmentof the hESCs to a substrate.
 5. The composition of claim 4, wherein thesubstrate is that from a tissue culture vessel.
 6. A method of culturingprimate embryonic stem cells in a feeder-free media comprising:culturing the primate embryonic stem cells in a culture medium which cansupport stem cells, the culture medium being essentially free of feedercells, and wherein the medium comprises human serum or a solubleattachment component thereof, thereby promoting undifferentiatedembryonic stem cell proliferation.
 7. The method of claim 6, wherein theculture medium comprises about 0.5% to about 2% human serum or a solubleattachment component thereof.
 8. The method of claim 7 furthercomprising enrichment of at least one embryonic stem cell solubleattachment component selected from the group consisting of 100 kDa, 150kDa, 200 kDa, 250 kDa, and 300kDa enriched by fractionation.
 9. Themethod of claim 8, wherein the enrichment is by fractionating the humanserum using a size-exclusion device.
 10. The method of claim 9, whereinthe size-exclusion device has a 300 KDa molecular weight cut-off. 11.The method of claim 9, wherein said size-exclusion device has a 100KDamolecular weight cut-off.
 12. The method of claim 9, wherein saidsize-exclusion device has a 30 KDa molecular weight cut-off.
 13. Themethod of claim 9, wherein the primate embryonic stem cells are humanembryonic stem cells (hESCs).
 14. A method of culturing primateembryonic stem cells in a feeder-free defined media comprising:culturing the primate embryonic stem cells in a culture medium which cansupport stem cells, the culture medium being substantially free offeeder cells and further comprising about 0.5% to about 2% human serumor a soluble attachment component thereof the culturing step beingconducted for over one month with the embryonic stem cells proliferatingin culture while maintaining the potential of the stem cells todifferentiate into derivatives of endoderm, mesoderm, and ectodermtissues, and while maintaining the karyotype of the stem cells.